Division of Infection and Immunity, International Institute for Zoonosis Control (Former Research Center for Zoonosis Control), Hokkaido University, Sapporo, Japan.
Graduate School of Infectious Diseases, School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
PLoS One. 2021 Oct 11;16(10):e0258317. doi: 10.1371/journal.pone.0258317. eCollection 2021.
Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.
炭疽是一种由革兰氏阳性芽孢形成细菌炭疽芽孢杆菌引起的人畜共患病。检测动物对炭疽亚致死暴露产生的天然抗体对于炭疽监测和有效控制措施至关重要。基于炭疽芽孢杆菌保护性抗原 (PA) 的血清学检测主要用于炭疽监测和疫苗评估。虽然该检测方法可靠，但由于 PA 与两种抗体均发生交叉反应，因此难以区分动物体内自然获得的抗体和疫苗诱导的免疫。尽管可以通过动物的疫苗接种史来解决这个问题，但在许多情况下，此类数据不易获取。在这项研究中，我们建立了一种针对炭疽芽孢杆菌荚膜生物合成蛋白 CapA 抗原的新酶联免疫吸附试验 (ELISA)，该方法对马疫苗诱导的抗体无交叉反应。通过计算机分析，我们筛选了编码在 pXO2 质粒上的编码序列，该质粒在兽医疫苗株 Sterne 34F2 中不存在，但在炭疽芽孢杆菌的毒力株中存在。在 8 个候选抗原中，荚膜生物合成蛋白 CapA (GBAA_RS28240) 和肽 ABC 转运体底物结合蛋白 (GBAA_RS28340) 被感染马血清中的抗体检测到。其中，据我们所知，CapA 尚未在其他研究中被鉴定为具有免疫反应性。考虑到炭疽芽孢杆菌的蛋白可溶性和特异性，我们制备了 CapA 的 C 末端区域，命名为 CapA322，并基于马模型开发了 CapA322-ELISA。CapA322-ELISA 与 PAD1-ELISA（ELISA 使用 PA 的结构域 1）的比较分析表明，CapA322-ELISA 可检测感染马血清中的抗 CapA 抗体，但对疫苗接种马血清无反应性。CapA322-ELISA 有助于在流行地区进行炭疽监测，本研究中鉴定的两种免疫反应性蛋白可以作为改进当前或未来疫苗开发的添加剂。
